Rat liver cytochrome P-450MC (hereinafter referred to as "P-450MC"), which is inducible by administration of 3-methylcholanthrene (hereinafter referred to as "MC"), is hemoprotein and has a monooxygenase activity towards external aliphatic compounds such as steroids or fatty acids through microsomal electron-transport chains.
Recently, there have been published reports on expression of heterogeneous genes in yeasts by using yeast alcohol dehydrogenase promoter or acidic phosphatase promoter. For example, expressions of a surface antigen of Hepatitis B virus and interferon were reported. Although the number of the reports on expression of heterogeneous genes in yeasts is smaller yet than that of the experiments using E. coli as a host, it is recently increasing.
With respect to P-450MC, whose molecular weight is as large as 59,300 daltons, which contains heme in its molecule and which has a high aryl hydrocarbon hydroxylase activity with a broad substrate spectrum, there has been, however, no report on the expression of its gene in yeasts.
The present inventors have extensively studied on the expression of P-450MC having such characteristics in yeasts, after succeeded in cloning of cDNA coding for P-450MC, constructed a plasmid carrying a ca. 2.7 Kb cDNA insert coding for P-450MC and isolated such plasmids. We reported that P-450MC is a protein consisting of 523 amino acid residues with a molecular weight of 59,300 daltons [Nucleic Acids Research, 12, 2929-2938 (1984)].
Based on these findings, the present inventions have further studied, and now succeeded in constructing a plasmid carrying a DNA fragment coding for P-450MC in such a form that the P-450MC coding region can easily be isolated therefrom as well as in construction of an expression plasmid which expresses the P-450MC gene in yeasts.
Moreover, the present inventors have now prepared transformant yeasts producing P-450MC by transforming yeasts with the said expression plasmids.
The transformant yeasts provided by the present invention produces P-450MC and have a monooxygenase activity which is derived from the P-450MC synthesized in the yeasts.
Thus provided transformant yeast cells, their microsomal fractions, which contain P-450MC, as well as P-450MC per se, which can be isolated therefrom, can be used for oxidation processes or oxidative disposal of organic compounds in industrial wastes.